摘要
通过引物设计,利用重叠延伸PCR(OE-PCR)和搭桥PCR方法扩增得到A型流感病毒M2蛋白缺失跨膜区基因以及HA多表位基因,分别克隆测序后以pET28a+为表达载体构建重组质粒pET28a+-M2d-HA。将重组质粒转化大肠杆菌BL21(DE3),筛选获得了高效表达流感病毒缺失跨膜区M2蛋白和HA多表位蛋白片段重组融合蛋白的工程菌,表达的重组蛋白约占菌体总蛋白的45%左右。表达的蛋白以包涵体形式存在,包涵体经亲和层析和阴离子交换层析纯化后复性,得到纯度在90%以上的目的蛋白。Westernblot结果显示纯化的重组蛋白具有较好的抗原性和特异性。为进一步研究广谱型流感病毒亚单位疫苗奠定了基础。
The influenza A virus matrix protein2 gene（M2） which deleted transmembrane region was amplified by overlap extending PCR, and the multi-epitope gene of hemagglutinin （HA） was PCR amplified with seven continuous synthesized segments by designing primer. The two gene segments were separately cloned into pMD18-T vector to sequence analysis and prokarytic expression vector pET28a ＋ to construct the recombinant plasmid pET28a ＋ -M2d-HA. The recombinant plasmid was transformed into E. coli BL21 （DE3）, and the high expression strain was obtained by screening monoelones. The recombinant protein existed as inclusion bodies, which accounted about 45% of the total cellular protein. The inclusion bodies were washed with 1% Triton X-100 solution twice, and dissolved in 8 moL/L urea solution. The solution protein was purified by Ni2^＋ affinity chromatography, and refolded by dilution renaturation, then purified by Q Sepharose FIr cation exchange column. The purity of the protein was over 90% by HPLC analysis. The result of Western blot showed it has good antigenicity and specificity. These results strongly supported for the further study of the broad-spectrum influenza virus subunit vaccine.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2007年第5期90-95,共6页
China Biotechnology
基金
云南省自然科学基金资助项目(2005C0062M)
关键词
流感病毒
亚单位疫苗
多表位
表达纯化
抗原性
Influenza virus Subunit vaccine Multiepitope Expression and purification Antigenicity
